CaCDPK15 positively regulates pepper responses to Ralstonia solanacearum inoculation and forms a positive-feedback loop with CaWRKY40 to amplify defense signaling

CaWRKY40 is a positive regulator of pepper (Capsicum annum) response to Ralstonia solanacearum inoculation (RSI), but the underlying mechanism remains largely unknown. Here, we functionally characterize CaCDPK15 in the defense signaling mediated by CaWRKY40. Pathogen-responsive TGA, W, and ERE boxes were identified in the CaCDPK15 promoter (pCaCDPK15), and pCaCDPK15-driven GUS expression was significantly enhanced in response to RSI and exogenously applied salicylic acid, methyl jasmonate, abscisic acid, and ethephon. Virus-induced gene silencing (VIGS) of CaCDPK15 significantly increased the susceptibility of pepper to RSI and downregulated the immunity-associated markers CaNPR1, CaPR1, and CaDEF1. By contrast, transient CaCDPK15 overexpression significantly activated hypersensitive response associated cell death, upregulated the immunity-associated marker genes, upregulated CaWRKY40 expression, and enriched CaWRKY40 at the promoters of its targets genes. Although CaCDPK15 failed to interact with CaWRKY40, the direct binding of CaWRKY40 to pCaCDPK15 was detected by chromatin immunoprecipitation, which was significantly potentiated by RSI in pepper plants. These combined results suggest that RSI in pepper induces CaCDPK15 and indirectly activates downstream CaWRKY40, which in turn potentiates CaCDPK15 expression. This positive-feedback loop would amplify defense signaling against RSI and efficiently activate strong plant immunity.


pCaCDPK15-driven GUS expression was upregulated by RSI and exogenously applied signaling mediators in pepper plants.
Our previous study performed a genome-wide identification of pepper CDPK members. A total of 35 CDPK genes were identified in the genome of the pepper variety CM334 using genome sequence data 38 (DOI:10.3389/fpls.2015.00737). The CaCDPK15 gene contained an N-variable domain, a protein kinase domain, an autoinhibitory domain, and a CaM-like domain, which are unique features of CDPK proteins 10 (Supplementary Fig. S1). CaCDPK15 exhibited inducible transcriptional expression in response to RSI (DOI:10.3389/fpls.2015.00737), suggesting a possible role in pepper immunity to R. solanacearum. In the present study, the CaCDPK15 promoter (pCaCDPK15) was identified, along with the cis-elements within the promoter (1 TCA element, 1 HSE, 1 ERE, and 7 W-boxes) (Fig. 1a). A pCaCDPK15-driven GUS reporter was expressed in pepper leaves by agroinfiltration, and GUS expression in pepper leaves was measured in response to RSI and exogenous application of salicylic acid (SA), methyl jasmonate (MeJA), abscisic acid (ABA), and ethephon (ETH). The results showed that GUS expression was upregulated by RSI, SA, MeJA, ABA, and ETH, with different temporal expression patterns (Fig. 1b).
CaCDPK15 is localized to the plasma membrane and nucleus. The subcellular localization of a protein can determine or influence its function. To determine the subcellular localization of CaCDPK15, we generated a CaCDPK15-GFP fusion construct driven by the constitutive CaMV35S promoter, and expressed the construct in Nicotiana benthamiana leaves by agroinfiltration. The subcellular locations of CaCDPK15-GFP and GFP control were visualized with laser scanning confocal microscopy. The results revealed that CaCDPK15-GFP Scientific RepoRts | 6:22439 | DOI: 10.1038/srep22439 was localized in both the plasma membrane and the nucleus, whereas the GFP control was localized in multiple subcellular compartments including the cytoplasm and the nucleus (Fig. 2).
Effect of CaCDPK15 silencing on pepper resistance to RSI. To test the role of CaCDPK15 in pepper immunity, we evaluated CaCDPK15 loss-of-function in pepper seedlings by performing virus-induced gene silencing (VIGS). The vectors TRV1 (PYL192) and TRV2:CaCDPK15 (PYL279) were separately transformed separately into Agrobacterium tumefaciens GV3101, and the two resulting GV3101 strains were mixed and co-infiltrated into pepper seedling leaves. The infiltrated seedlings were incubated at 16 °C for 56 h without light, and then were kept at 25 °C. Six independent experiments were performed, and we obtained approximately 100 plants of TRV:00 and 100 plants of TRV:CaCDPK15. A TRV:PDS control construct was used in the same way to monitor gene silencing by the resulting photobleaching phenotype 36 . Six plants were randomly selected to check the gene silencing efficiency. In TRV:CaCDPK15 pepper plants, CaCDPK15 transcript levels were reduced to ~30% of those in TRV:00 plants (Fig. 3a). The R. solanacearum strain FJC100301 was used to inoculate six individual TRV:CaCDPK15 plants and six individual TRV:00 empty vector control plants. We stained R. solanacearum-infected CaCDPK15-silenced and control leaves with DAB (indicator of H 2 O 2 accumulation) and trypan blue (indicator of cell death or necrosis). Strongly polymerized DAB (dark brown) and hypersensitive response (HR)-mimicking cell death were detected in control leaves at 48 h post inoculation (hpi), whereas the intensities of DAB and trypan blue staining were distinctly reduced in CaCDPK15-silenced leaves (Fig. 3b). Our data also showed that R. solanacearum growth was significantly increased in CaCDPK15-silenced pepper plants, manifested by higher colony-forming units (cfu) compared with that in control plants (Fig. 3c). The expression of known pepper defense genes involved in the response to pathogen infection was analyzed by quantitative real-time PCR (qPCR) analysis. The results showed that transcript levels of the defense-related pepper genes CaPR1 39 , CaDEF1 40 , CaPO2 41 , and CaHIR1 42 were lower in CaCDPK15-silenced leaves than in leaves of control pepper plants at 24 hpi (Fig. 3d). At 14 days post inoculation (dpi), we observed definite wilting symptoms on TRV:CaCDPK15 pepper leaves, but the TRV:00 empty-vector control leaves exhibited only faint wilting symptoms (Fig. 3e).
Transient CaCDPK15 expression induces the hypersensitive response, cell death, and H 2 O 2 accumulation in pepper leaves. We attempted to generate transgenic CaCDPK15-overexpressing tobacco plants, but found that CaCDPK15 overexpression was lethal in transgenic tobacco. Therefore, a transient cis-elements including one TCA-element, one HSE, one ERE and seven W-boxes in pCaCDPK15. (b) The pCaCDPK15 driven GUS expression was promoted by exogenous application of SA, MeJA, ETH and ABA and RSI. The leaves of pepper plants at eighty-leaf stage ware infiltrated with GV3101 cells (OD 600 = 0.6) containing pCabZIP63:GUS, and 24 hours later the plants were treated with 1 mm SA, 100 μm MeJA, 100 μm ETH, 100 μm ABA, or inoculated with the R. solanacearum (OD 600 = 0.6). The leaves were harvested at different time points for GUS activity assay by microplate reader using pepper leaves treated with ddH 2 O as mock. Data are the means ± SD from at least three independent experiments. Asterisks indicate statistically significant differences compared with Mock (treated with ddH 2 O). (t-test, **P < 0.01).
overexpression system for CaCDPK15 was generated by agroinfiltration of 35S:CaCDPK15 or 35S:00 (empty vector) into pepper leaves (Fig. 4a). HR-mediated cell death and H 2 O 2 accumulation were assessed by staining with trypan blue to identify necrotic cells and DAB, respectively. The 35S:CDPK15 construct distinctly induced a necrotic response in pepper leaves and H 2 O 2 accumulation, whereas the empty-vector control did not induce a necrotic response and resulted in only weak DAB staining. We also performed an ion leakage test to analyze the severity of plasma membrane damage and thereby the severity of cell necrosis in cells transiently expressing CaCDPK15. Pepper leaves transiently overexpressing CaCDPK15 exhibited more ion leakage at 24 and 48 h after agroinfiltration than that in leaves expressing the empty vector control (Fig. 4b). Real-time RT-PCR analysis of CaCDPK15 transcripts in the transient expression system showed that transcripts were higher in leaves expressing 35 S:CaCDPK15 than in empty-vector control leaves (Fig. 4c). We also examined changes in the expression of defense-related genes including SA-responsive CaPR1 and CaNPR1 43 , JA-responsive CaDEF1, and CaWRKY40 in the transient expression system. The results showed that the relative transcript levels of CaPR1, CaNPR1, CaDEF1, and CaWRKY40 increased continuously during transient expression of CaCDPK15.
The effect of transient overexpression of CaCDPK15 on the binding of CaWRKY40 to its target genes. CaCDPK15 may modify CaWRKY40 transcriptional activity by altering its binding to the promoters of target genes. We tested this hypothesis by performing chromatin immunoprecipitation (ChIP) experiments. A specific primer pair was designed based on the flanking sequences of each typical W-box in the promoters of CaPR1, CaNPR1, and CaDEF1 (Fig. 5a). For promoters with more than one W-box, the primer pairs were screened for product amplification and used in the real-time RT-PCR measurements of specific CaWRKY40 binding to the promoter. The results showed that CaWRKY40 binding to the promoters of CaPR1, CaNPR1, and CaDEF1 was significantly enhanced by transient CaCDPK15 overexpression (Fig. 5b,c).
Detection of potential interactions between CaCDPK15 and CaWRKY40 by co-immunoprecipitation analysis. If CaCDPK15 acts as an upstream modifier of CaWRKY40 signaling, one possibility might be that CaWRKY40 is a target of CaCDPK15. We tested this hypothesis by performing co-immunoprecipitation (co-IP) analyses to evaluate possible interactions between the two proteins. These experiments employed a transient coexpression system in N. benthamiana leaves with the tagged constructs 35S:CaCDPK15-HA and 35S:CaWRKY40-Flag, and the positive control constructs 35S:CaPIK1-Flag and 35S:CaSGT1-HA 44 . The results showed that CaWRKY40 does not interact with CaCDPK15, indicating that CaWRKY40 is not a direct target of CaCDPK15 ( Supplementary Fig. S2). ChIP analysis of CaWRKY40 binding to the CaCDPK15 promoter. As CaWRKY40 significantly activated CaCDPK15 expression, we speculated that CaWRKY40 might act as a TF in directly modulating CaCDPK15 expression. We tested this hypothesis by performing ChIP to determine if CaWRKY40 binds to the CaCDPK15 promoter. For this experiment, GV3101 cells containing the p35S:CaWRKY40-HA construct or the empty vector were infiltrated into pepper (GZ03) leaves, which were harvested at 48 hpi for chromatin isolation. The isolated chromatin was randomly sheared into fragments with lengths of 300− 500 base pairs, and chromatin fragments that bound to CaWRKY40 were immunoprecipitated using the HA antibody. The resulting DNA fragments were isolated and used as templates for PCR analysis with specific primer pairs. The results showed that only the primer pairs flanking the fifth and sixth W-boxes produced amplified products, suggesting that CaWRKY40 directly binds to the CaCDPK15 promoter (Fig. 7a). To test the effect of RSI on CaWRKY40 binding to the CaCDPK15 promoter, ChIP analysis was performed during RSI. The real-time RT-PCR results showed that the infected pepper leaves had higher enrichment of CaWKRY40 in the CaCDPK15 promoter compared with that of the mock control (Fig. 7b).

Discussion
Although CDPKs and WRKYs have both been implicated in pathogen attack 45 , the molecular linkage between these two proteins has not been established. We provide strong evidence that CaCDPK15 forms a positive-feedback loop with CaWRKY40 during RSI in pepper, and previously established that CaWRKY40 is a positive regulator of pepper's response to RSI 36 .
Accumulating evidence indicates that upregulated genes responding to plant defense signaling can have important roles in disease resistance 46 . The CaCDPK15 promoter contains the following cis-elements: 1 SA-responsive TCA element, 1 ethylene-responsive ERE box, and 7W-boxes. These cis elements are frequently involved in plant immunity responses 47 . Transgenic tobacco expressing pCaCDPK15:GUS consistently exhibited significantly inducible GUS expression in response to RSI, suggesting that CaCDPK15 might act as a positive regulator in pepper's response to RSI. This possibility was confirmed by loss-of-function experiments, which showed that CaCDPK15 silencing significantly reduced pepper resistance to RSI, and significantly down-regulated the expression of the immunity marker genes CaNPR1, CaPR1, and CaDEF1. By contrast, transient CaCDPK15 overexpression in pepper plants triggered HR-mimicking cell death, enhanced electrolyte leakage, and enhanced accumulation of H 2 O 2 . Ca 2+ is a ubiquitous signal in plant defense responses to biotic and abiotic stresses 48,49 , and CDPKs are one of the Ca 2+ sensors that relay Ca 2+ signatures to downstream components via protein phosphorylation and transcriptional reprogramming 45 . These combined results suggest that CaCDPK15 acts as a positive regulator of pepper's response to RSI.
Our previous work showed that CaWRKY40 was upregulated in response to RSI and high temperature/high humidity, and functioned as a positive regulator in pepper's response to these two stresses 36 . CaWRKY40 and CaCDPK15 have similar expression patterns and functions in pepper's response to RSI, which suggests a close relationship between these two genes and their encoded proteins. This was corroborated by data showing that the transcriptional expression of CaWRKY40 was upregulated by transient CaCDPK15 overexpression in pepper plants, and was significantly down-regulated by VIGS-mediated CaCDPK15 silencing. Ca 2+ influx occurs very early during stress challenge. CDPK proteins localized in the plasma membrane and/or cytoplasm are expected to be involved in early signaling pathways that respond to biotic and abiotic stresses 49 . CaCDPK15 is believed to act as an upstream regulator of CaWRKY40. Similar molecular linkages between CDPKs and WRKYs have been reported previously 50,51 . For example, the overexpression of wheat (Triticum aestivum) TaCPK2-A in rice (Oryza sativa) promoted OsWRKY45-1 expression, which is a TF involved in resistance to fungi and bacteria, by regulating genes involved in JA and SA signaling 50 . In Arabidopsis, the CDPK4/5/6/11 isoforms phosphorylate a specific subgroup of WRKY TFs (WRKY8/28/48) that regulate crucial transcriptional reprogramming that restricts pathogen growth 51 . However, unlike these CDPK and WRKY proteins, this study provided evidence that CaCDPK15 and CaWRKY40 do not directly interact with each other, and CaCDPK15 does not phosphorylate CaWRKY40; co-IP detected no interaction between CaCDPK15 and CaWRKY40, and CaCDPK15 localized to nuclei. Therefore, CaCDPK15 might phosphorylate and activate other TFs that target CaWRKY40. Further identification of possible CaCDPK15 interactors that subsequently target CaWRKY40 might provide new insights into the mechanism of pepper immunity mediated by CaCDPK15 and CaWRKY40.
We showed that CaCDPK15 modulates CaWRKY40 expression. Unexpectedly, we found that CaCDPK15 expression in pepper plants was transcriptionally upregulated by transient CaWRKY40 overexpression, whereas it was downregulated by transient overexpression of CaWRKY40-SRDX (repressor) and by CaWRKY40 silencing. This suggests that there is a positive-feedback loop between CaCDPK15 and CaWRKY40. The presence of 7W-boxes in the CaCDPK15 promoter indicated that WRKY TFs might directly transcriptionally regulate CaCDPK15 expression. Our ChIP analysis data revealed that CaWRKY40 binds to the CaCDPK15 promoter, which was significantly enhanced by RSI. These results strongly suggest that CaWRKY40 acts as a direct TF in the transcriptional modulation of CaCDPK15 expression. Similar positive-feedback loops in plant responses to stresses including pathogen attack have been reported [52][53][54] . For example, SA was reported to act in a positive-feedback loop with ACCELERATED CELL DEATH6 (ACD6) to potentiate plant CaWRKY40-HA and that containing 35S:CaCDPK15-Flag were mixed at a ratio of 1:1 and were co-infiltrated into pepper leaves, with GV3101 cells containing 35S:00 as mock. The leaves were harvested at 48 hpi for chromatin preparation, the isolated chromatins were digested with micrococcal nuclease and the acquired DNA collections with 300-500 bp in length were used as templates for real-time RT-PCR to assay the bindings of CaWRKY40 to the promoters of its target genes, for each target gene of CaWRKY40, a specific primer pair flanking each typical W-box was designed and the one (primer pair based on 1 W in CaPR1 promoter, 1 W in CaNPR1 promoter and 2 W in CaDEF1 promoter, respectively) that amplified product was used in the real-time RT-PCR analysis. Relative enrichment levels of samples of the CaWRKY40 transient overexpression were set to 1 after normalization by input. (a,b) Data are the means ± SD from at least three independent experiments. Asterisks indicate statistically significant differences compared with 35S:00 (EV) and 35S:CaWRKY40/35S:00 (EV). (t-test, **P < 0.01).
Scientific RepoRts | 6:22439 | DOI: 10.1038/srep22439 responsiveness to pathogen-associated molecular patterns (PAMPs) 52 ; HEAT SHOCK PROTEIN101 and HEAT STRESS-ASSOCIATED 32-KD PROTEIN form a positive-feedback loop that modulates long-term acquired thermotolerance 53 ; and positive-feedback regulation by ABA on LOS6/ABA1 expression provides a quick adaptation strategy for plants under osmotic stress 54 . In general, plant defense systems tend to focus on early stress-mediated events, and these positive-feedback loops may be important for amplifying defense signaling 55 . Our data also showed that exogenous application of SA, MeJA, ABA, and ETH synergistically upregulated  CaWRKY40-HA was inoculated to the pepper leaves, which are harvested at 48 hpi for preparation of chromatin for ChIP assay, the immunoprecipitated DNA was used as template for PCR with specific primer pairs designed according to the seven W-boxes. Lanes 1, input (total DNA-protein complex); lanes 2, (DNAprotein complex) immunoprecipitated with anti-HA antibody (α -HA), the anti-Flag antibody (α -Flag) was used as a negative control to discriminate the possible unspecific IP in HA-IgG. (b) The binding of CaWRKY40 to the promoter of CaCDPK15 was enhanced by RSI. GV3101 cells containing the construct of 35S:CaWRKY40-HA was inoculated to the pepper leaves, 24 hours later, the leaves were further inoculated with R. solanacearum, 24 hours later, the leaves were harvested for preparation of chromatin for ChIP assay, and a specific primer pair was used in the real-time RT-PCR. Data are the means ± SD from at least three independent experiments. Asterisks indicate statistically significant differences compared with the treatment of MgCl 2 (Mock, [b]). (t-test, **P < 0.01).
Scientific RepoRts | 6:22439 | DOI: 10.1038/srep22439 CaCDPK15 expression, which is consistent with their effects on CaWRKY40 expression 36 . These results strongly suggest a molecular linkage between CaCDPK15 and CaWRKY40.
In our previous study, CaWRKY40 positively regulated pepper response to RSI and plant thermotolerance under high humidity, which is important for plant adaption to conditions that promote the invasion and growth of soil-borne pathogens. Although the present study focused on the role of CaCDPK15 in pepper immunity, there were indications that CaCDPK15 might be involved in thermotolerance under high humidity. For example, we identified an HSE element in the CaCDPK15 promoter, determined that pCaCDPK15-driven GUS expression also was activated by heat stress treatment. and found that CaCDPK15 silencing impaired plant thermotolerance and downregulated the expression of the thermotolerance-associated marker gene CaHSP24. CaHSP24 expression was consistently upregulated by CaCDPK15 expression (data not shown).
Collectively, our data indicate that CaCDPK15 expression is upregulated by RSI, which indirectly activates the transcriptional expression of downstream CaWRKY40. Likewise, transcriptional expression of CaWRKY40 directly activates the transcriptional expression of CaCDPK15. This generates a positive-feedback loop that would rapidly amplify plant signaling in response to RSI and efficiently activate plant defense responses. Histochemical staining. Staining with trypan blue and DAB was performed according to the previously published method of Choi et al. 56 . For trypan blue staining, pepper leaves were boiled in trypan blue staining solution for 2 min, left at room temperature for 8 h, transferred into a chloral hydrate solution (2.5 g of chloral hydrate dissolved in 1 mL of distilled water), and boiled for 20 min to destain. After multiple changes of chloral hydrate solution to reduce the background, samples were mounted in 70% glycerol. For DAB staining, the leaves were stained overnight in 1 mg ml −1 DAB. The stained leaves were cleared by boiling in lactic acid:glycerol:absolute ethanol [1:1:3 (v/v/v)], and then destained overnight in absolute ethanol. Representative images of DAB and trypan blue staining were photographed with a light microscope (Leica, Wetzlar, Germany).

Virus-induced gene silencing (VIGS) of CaCDPK15 in pepper plants. The tobacco rattle virus
(TRV)-based VIGS system was employed to silence CaCDPK15. The PYL192 and PYL279 VIGS vectors were described previously 57 . A fragment of the transcribed region of CaCDPK15 was amplified using gene-specific primers (5ʹ -GGGGACAAGTTTGTACAAAAAAGCAGGCTTCTTTTCTTTTCGC CCTTTA-3ʹ and 5ʹ -GGGGACCACTTTGTACAAGAAAGCTGGGTCAATGAACT CCATCCAGCA-3ʹ ), and cloned into the PYL279 VIGS vector using the Gateway cloning system (Invitrogen). The PYL192 and PYL279 vectors were with or without CaCDPK15, respectively. PYL279 contained a 250-or 500-bp PDS fragment. The PYL192 and PYL279 vectors were transformed into A. tumefaciens strain GV3101. Agrobacterium harboring PYL192 with PYL279 (PYL192 vector with PYL279 as TRV:00), PYL279-CaCDPK15 (PYL192 with PYL279-CaCDPK15 as TRV:CaCDPK15), or PYL279-PDS (OD 600 = 1.0) were mixed at a 1:1 ratio, and the mixture was infiltrated into cotyledons of 2-week-old pepper plants using a 1 ml sterile syringe without a needle. The Agrobacterium-inoculated pepper plants were grown for 2-3 weeks in a growth chamber at 16 °C (in darkness for the first 56 h) with 45% relative humidity, and then transferred into a growth room at 25 ± 2 °C, 60-70 μmol photons m −2 s −1 , 70% relative humidity, and a 16-h light/8-h dark photoperiod. Transient expression assay of CaCDPK15. Agrobacterium tumefaciens strain GV3101 harboring the pK7WG2-CaCDPK15 vector was cultured to OD 600 = 1.0 in induction medium (10 mM ethanesulfonic acid, pH 5.7, 10 mM MgCl 2 , and 200 mM acetosyringone) and diluted to OD 600 = 0.8. The diluted culture was injected into pepper or Nicotiana benthamiana leaves using a syringe without a needle. The plants were kept in a growth room for 2 days, and then the injected leaves were harvested for further use.

Quantitative real-time PCR.
To determine the relative transcript levels of selected genes, real-time PCR was performed with specific primers (Supplementary Table S1 and Table S2) according to the manufacturer's instructions for the BIO-RAD Real-time PCR system (Foster City, CA, USA) and the SYBR Premix Ex Taq II system (TaKaRa Perfect Real Time). Total RNA was isolated from pepper plants using TRIzol reagent (Invitrogen), and was reverse-transcribed using the PrimeScript RT-PCR kit (TaKaRa, Dalian, China) 59 . A 10-fold dilution of the resulting cDNA was amplified using the SYBR Premix Ex Taq II kit and the BIO-RAD Real-time PCR system in a 10 μl volume with the following program: 95 °C for 30 s; 40 cycles of 95 °C for 5 s and 60 °C for 34 s; and 95 °C for 15 s. Amplification of the target genes was monitored every cycle by SYBR green fluorescence. The Ct (threshold cycle) value, which is defined as the real-time PCR cycle at which a statistically significant increase of reporter fluorescence was first detected, was used as a measure for the starting target gene copy number. Three replicates of each experiment were performed. Data were analyzed by the Livak method and expressed as a normalized relative expression level (2 −ΔΔCT ) of the respective genes. The relative transcript levels were normalized with respect to the transcript levels of CaActin and 18 s rRNA. In each case, three technical replicates were performed for at least three independent biological replicates.
Chromatin immunoprecipitation analysis. The 35S:CaWRKY40-HA and 35S:CaCDPK15-Flag constructs were generated by Gateway cloning (Invitrogen) 60 , and were transformed into Agrobacterium strain GV3101. GV3101 cells containing 35S:CaWRKY40-HA and 35S:CaCDPK15-Flag were co-infiltrated at a ratio of 1:1 or infiltrated individually into pepper leaves, which were harvested at 48 hpi for chromatin preparation. ChIP was performed according to standard protocols. Briefly, approximately 2 g of pepper leaves was treated with either 10 mM bithionol sulfoxide or DMSO (solvent control) for 16 h and subsequently fixed with 1.0% formaldehyde for 5 min. Antibody against HA or FLAG (Santa Cruz Biotechnology) were used for immunoprecipitation. Protein-A-agarose beads were blocked with salmon sperm DNA and used to pull down the protein-DNA complex. Equal amounts of starting plant material and the ChIP products were used for PCR or real-time PCR with specific primers for the promoters of CaCDPK15, CaPR1, CaNPR1, and CaDEF1 (Supplementary Table S3).